Chasing a ghost?--Issues with the determination of circulating levels of endotoxin in human blood.

Reliable quantification of bacterial products such as endotoxin is important for the diagnosis of Gram-negative infection and for the monitoring of its treatment. Further, it is important to identify patients with persistent subclinical level of bacterial products in their systemic circulation as data from animal studies also suggest this may be correlated with the onset of metabolic syndrome. In this review, we first aim to describe the principles of the Limulus amoebocyte lysate (LAL) test, an assay that is used to quantify endotoxin, and the various shortcomings that must be addressed before it can become a reliable means of quantifying endotoxin in samples derived from blood. We then review published data regarding endotoxin levels in healthy subjects and those with sepsis, inflammatory bowel disease, liver disorders and metabolic disorders such as obesity and diabetes. We also review the evidence regarding influence of macronutrients in augmenting the levels of systemic endotoxin. The results of this review show that reported mean levels of endotoxin in the systemic circulation of healthy humans and of those with various clinical disorders vary over a wide range. Further, this review shows that a significant proportion of this variation can be related to the method that was used to prepare plasma and serum samples prior to assay and its ability to reduce the effect of various blood borne factors that interfere with the LAL assay.

The Characteristics and Function of Bacterial Lipopolysaccharides and Their Endotoxic Potential in Humans.

Cross-talk between enteral microbiota and human host is essential for the development and maintenance of the human gastrointestinal and systemic immune systems. The presence of lipopolysaccharides (LPS) lysed from the cell membrane of Gram-negative bacteria in the gut lumen is thought to promote the development of a balanced gut immune response whilst the entry of the same LPS into systemic circulation may lead to a deleterious pro-inflammatory systemic immune response. Recent data suggest that chronically low levels of circulating LPS may be associated with the development of metabolic diseases such as insulin resistance, type 2 diabetes, atherosclerosis and cardiovascular disease. This review focuses on the cross-talk between enteral commensal bacteria and the human immune system via LPS. We explain the structural characterisation of the LPS molecule and its function in the bacteria. We then examine how LPS is recognised by various elements of the human immune system and the signalling pathways that are activated by the structure of the LPS molecule and the effect of various concentrations. Further, we discuss the sequelae of this signalling in the gut-associated and systemic immune systems i.e. the neutralisation of LPS and the development of tolerance to LPS.

Aspirin-induced increase in intestinal paracellular permeability does not affect the levels of LPS in venous blood of healthy women.

The presence of subclinical levels of LPS from Gram-negative bacteria, also referred to as endotoxin, in the circulation may induce a pro-inflammatory immune response that leads to the development of obesity and insulin resistance. Recent data indicate that high-fat meals may elevate circulating levels of LPS. However, it is currently unclear how the LPS transits from the gut lumen to the general circulation. We determined whether aspirin-induced damage of the small intestinal mucosa, evidenced by an increase in the paracellular permeability, allows greater transit of LPS into the systemic circulation. The 3-h cumulative excretion of lactulose was significantly increased after the consumption of aspirin solution relative to that after the consumption of an equal volume of water in 15 healthy women (median after aspirin 0.09% of dose vs. median after water 0.03% of dose; P = 0.004). Dosage with aspirin also significantly increased the lactulose : mannitol ratio (median after aspirin 0.014 vs. median after water 0.005; P = 0.017). However, serum LPS levels after the consumption of the aspirin solution were not significantly different from those after consumption of the control (plain water). Further, there was no correlation between body fat content and circulating levels of LPS.

The Limulus Amebocyte Lysate assay may be unsuitable for detecting endotoxin in blood of healthy female subjects.

We examined the factors that may influence the outcome of the Limulus Amebocyte Lysate (LAL) assay, when it is used for quantifying Gram-negative bacterial endotoxin, also referred to as lipopolysaccharide (LPS), in samples of human blood. We found that the method recommended by the manufacturers, based on the reaction time, was inaccurate with any type of serum samples due to the slowing of the initial phase of reaction, likely by serum proteins. We describe an alternative method that is more accurate for use with heated serum samples. Further, we found that components of fresh serum irreversibly sequester endotoxin but that this action may be largely prevented by dilution and heating, but only if this occurs prior to the addition of endotoxin. The tests also indicated that a number of types of proprietary plastic vacutainers appeared to contain significant amounts of endotoxin. However, even when appropriate blood collection containers and calculation methods were used, the levels of endotoxin in serum samples detected by LAL assay were unlikely to reflect the total quantities of endotoxin in that sample and more likely to reflect the capacity of a given serum sample to sequester endotoxin.

Epigenetic control of estrogen receptor expression and tumor suppressor genes is modulated by bioactive food compounds.

BACKGROUND: The tumor suppressor genes p15(INK)4(b) and p16(INK)4(a) as well as the estrogen receptor-α (ESR1) gene are abnormally methylated and expressed in colon cancer. The cancer-preventative abilities of several bioactive food components have been linked to their estrogenic and epigenetic activities. METHODS: The effect of folic acid, zebularine, resveratrol, genistein and epigallocatechin-3-gallate (EGCG) on tumor cell growth, promoter methylation of ESR1, p15(INK)4(b) and p16(INK)4(a) and gene expression of ESR1 and ESR2 was analyzed in Caco-2 cells. Gene expression was measured using real-time PCR, and promoter CpG methylation was assessed using bisulfite conversion and methylation-specific PCR. RESULTS: After exposure to a high concentration of folic acid (20 µmol/l), enhanced cancer cell growth and concomitant increased methylation of the ESR1 (3.6-fold), p16(INK)4(a) and p15(INK)4(b) promoters was observed. A lower concentration of folic acid (2 µmol/l) decreased cell growth. The phytoestrogens genistein and resveratrol enhanced expression of ESR1 (genistein 200 µmol/l: 2.1-fold; resveratrol 50 µmol/l: 6.3-fold) and ESR2 (2.6- and 3.6-fold, respectively). Genistein and resveratrol treatment increased promoter methylation of ESR1 (genistein 200 µmol/l: 2.9-fold; resveratrol 50 µmol/l: 1.4-fold). For p16(INK)4(a), increased methylation was found after exposure to 10 µmol/l resveratrol, but for p15(INK)4(b), decreased methylation was found. Both components showed growth-inhibitory activities. For EGCG, growth inhibition at 100 µmol/l and suppressed promoter methylation of tumor suppressor genes (p16(INK)4(a): 0.9-fold; p15(INK)4(b): 0.6-fold) was seen. CONCLUSIONS: Our results show that these food compounds regulate ESR and tumor suppressor gene expression by multiple mechanisms including epigenetic processes. An improved understanding of these epigenetic effects could therefore support specific dietary concepts of epigenetic cancer prevention and intervention.